We study cellular and molecular mechanisms involved in development of neurons of the central nervous system and the formation of central synapses, utilizing cell culture, microscopy and molecular techniques. Agrin, a proteoglycan secreted by motoneurons, is required for postsynaptic differentiation in skeletal muscle. A transmembrane form of agrin is widely expressed in the central nervous system but its functions are not well understood, although there is evidence for its involvement in neurite outgrowth as well as synapse formation and function. Our current studies focus on the functions and trafficking of transmembrane agrin in hippocampal neurons. We previously used siRNA suppression of agrin expression to demonstrate that endogenous agrin positively regulates the filopodia of developing hippocampal neurons in culture. We now have used time-lapse studies of living neurons to show that agrin regulates filopodia by increasing their stability and rate of initiation. We previously found that the expression of the transmembrane form of agrin in skeletal muscle and other cultured cells, including hippocampal neurons, induces the formation of filopodia and that the N-terminal extracellular moiety of transmembrane agrin is necessary and sufficient to do this in cell lines. We now have shown the importance of this domain in the induction of filopodia in hippocampal neurons. Moreover we have shown that the glycosaminoglycan side chains, which are attached to the N-terminal moiety, play an important role in filopodia induction in both cell lines and neurons. We have begun to investigate the roles of Rho-family GTP-binding proteins in this induction of filopodia and have found that transfection with agrin results in a striking increase in the activation of Cdc42. In order to study agrin trafficking in living neurons, we have created new transmembrane agrin plasmids that are more highly expressed than the previous ones. In addition, we have created transmembrane agrin plasmids containing a tetracysteine motif that will allow sequential labeling of previously existing and newly-synthesized agrin with different fluorescent dyes. This will facilitate studies of intracellular trafficking and turnover.